The development of an effective HIV vaccine is perhaps the greatest challenge currently facing the biomedical research community. A consensus is emerging that an effective vaccine must activate both the humoral and cellular arms of the immune response and, in addition, take into account the tremendous variability among HIV isolates. The development and refinement of such a vaccine is crucially dependent on model systems in which specific B and T cell responses can be precisely quantified. Our preliminary studies show that a single HIV envelope elicits strong antibody responses in mice when administered first as DNA, then as recombinant vaccinia virus, and finally as purified protein (i.e., a D-V-P vaccination regimen). The overall aim of this project is to use a mouse model to comprehensively evaluate the D-V-P as a vehicle for the delivery of multiple HIV envelopes in a highly immunogenic form. We will apply methodologies that are well-established in our laboratory to measure envelope-specific B cell and CD4+ and CD9+ T cell responses at the single cell level. Initially, we will characterize specific cellular responses during the immunization of mice with single HIV envelopes using the D-V-P regimen. Two distinct, well characterized envelopes from the HIV-1 strains IIIB and MN will be used. The second part of this project will test the effectiveness of the D-V-P regimen in presenting multiple envelopes simultaneously to elicit a greater diversity of B and T cell responses. In the first experiments in this series, both IIIB and MN will be included in each step of the D-V-P regimen. We will then address the immunological consequences of administering IIIB or MN at only one or two of the vaccination steps. Finally, we will evaluate the response of mice to a proposed multi-envelope clinical vaccine based on the D-V-P regimen. These studies will provide information that is essential for the rational evaluation and refinement of multi-step, multi-vector vaccination strategies.